Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner

2021 ◽  
Vol 478 (1) ◽  
pp. 197-215 ◽  
Author(s):  
Yuning Wang ◽  
Roya Tadayon ◽  
Liliana Santamaria ◽  
Pascal Mercier ◽  
Chantal J. Forristal ◽  
...  

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A–C2G, 13%–33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a ‘calcium-catching’ mechanism to respond to calcium influx during membrane repair.

2020 ◽  
Vol 219 (5) ◽  
Author(s):  
Adam Horn ◽  
Shreya Raavicharla ◽  
Sonna Shah ◽  
Dan Cox ◽  
Jyoti K. Jaiswal

Plasma membrane injury can cause lethal influx of calcium, but cells survive by mounting a polarized repair response targeted to the wound site. Mitochondrial signaling within seconds after injury enables this response. However, as mitochondria are distributed throughout the cell in an interconnected network, it is unclear how they generate a spatially restricted signal to repair the plasma membrane wound. Here we show that calcium influx and Drp1-mediated, rapid mitochondrial fission at the injury site help polarize the repair response. Fission of injury-proximal mitochondria allows for greater amplitude and duration of calcium increase in these mitochondria, allowing them to generate local redox signaling required for plasma membrane repair. Drp1 knockout cells and patient cells lacking the Drp1 adaptor protein MiD49 fail to undergo injury-triggered mitochondrial fission, preventing polarized mitochondrial calcium increase and plasma membrane repair. Although mitochondrial fission is considered to be an indicator of cell damage and death, our findings identify that mitochondrial fission generates localized signaling required for cell survival.


2021 ◽  
Vol 220 (5) ◽  
Author(s):  
Goutam Chandra ◽  
Sen Chandra Sreetama ◽  
Davi A.G. Mázala ◽  
Karine Charton ◽  
Jack H. VanderMeulen ◽  
...  

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
David Y. Barefield ◽  
Jordan J. Sell ◽  
Ibrahim Tahtah ◽  
Samuel D. Kearns ◽  
Elizabeth M. McNally ◽  
...  

AbstractMuscular dystrophies are disorders characterized by progressive muscle loss and weakness that are both genotypically and phenotypically heterogenous. Progression of muscle disease arises from impaired regeneration, plasma membrane instability, defective membrane repair, and calcium mishandling. The ferlin protein family, including dysferlin and myoferlin, are calcium-binding, membrane-associated proteins that regulate membrane fusion, trafficking, and tubule formation. Mice lacking dysferlin (Dysf), myoferlin (Myof), and both dysferlin and myoferlin (Fer) on an isogenic inbred 129 background were previously demonstrated that loss of both dysferlin and myoferlin resulted in more severe muscle disease than loss of either gene alone. Furthermore, Fer mice had disordered triad organization with visibly malformed transverse tubules and sarcoplasmic reticulum, suggesting distinct roles of dysferlin and myoferlin. To assess the physiological role of disorganized triads, we now assessed excitation contraction (EC) coupling in these models. We identified differential abnormalities in EC coupling and ryanodine receptor disruption in flexor digitorum brevis myofibers isolated from ferlin mutant mice. We found that loss of dysferlin alone preserved sensitivity for EC coupling and was associated with larger ryanodine receptor clusters compared to wildtype myofibers. Loss of myoferlin alone or together with a loss of dysferlin reduced sensitivity for EC coupling, and produced disorganized and smaller ryanodine receptor cluster size compared to wildtype myofibers. These data reveal impaired EC coupling in Myof and Fer myofibers and slightly potentiated EC coupling in Dysf myofibers. Despite high homology, dysferlin and myoferlin have differential roles in regulating sarcotubular formation and maintenance resulting in unique impairments in calcium handling properties.


2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Catarina Dias ◽  
Jesper Nylandsted

AbstractMaintenance of plasma membrane integrity is essential for normal cell viability and function. Thus, robust membrane repair mechanisms have evolved to counteract the eminent threat of a torn plasma membrane. Different repair mechanisms and the bio-physical parameters required for efficient repair are now emerging from different research groups. However, less is known about when these mechanisms come into play. This review focuses on the existence of membrane disruptions and repair mechanisms in both physiological and pathological conditions, and across multiple cell types, albeit to different degrees. Fundamentally, irrespective of the source of membrane disruption, aberrant calcium influx is the common stimulus that activates the membrane repair response. Inadequate repair responses can tip the balance between physiology and pathology, highlighting the significance of plasma membrane integrity. For example, an over-activated repair response can promote cancer invasion, while the inability to efficiently repair membrane can drive neurodegeneration and muscular dystrophies. The interdisciplinary view explored here emphasises the widespread potential of targeting plasma membrane repair mechanisms for therapeutic purposes.


1981 ◽  
Author(s):  
C Rupp ◽  
C Kuyas ◽  
A Häberli ◽  
M Furlan ◽  
E A Beck

Inherited hypodysfibrinogenemia (fibrinogen Bern I) was found in four members (two generations) of a family with no haemorrhagic or thrombotic history. Fibrin aggregation curves (350 nm, 37°C) with patient plasma or purified fibrinogen Bern I, after addition of thrombin, were normal at high calcium concentrations (5mM) but delayed at lower calcium concentrations (≤0.lmM). The release of fibrinopeptide A was normal. Whereas the polypeptide chains of fibrinogen Bern I were indistinguishable from normal fibrinogen by SDS-gel-electrophoresis, an abnormal γ-chain with a decreased negative charge was found by isoelectric focussing.Plasmic degradation o| normal fibrinogen, in the presence of calcium (≥ImM), results in only one terminal D fragment which is stabilized by calcium against further degradation of γ-chains. In contrast, degradation of fibrinogen Bern I, under the same conditions, yielded at least two additional smaller D fragments. In conclusion, fibrinogen Bern I is characterized by defective calcium binding in the D domain of the γ-chain.


2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Jestine Philip

Purpose Emerging technologies are capable of enhancing organizational- and individual-level outcomes. The organizational behavior (OB) field is beginning to pursue opportunities for researching emerging technologies. This study aims to describe a framework consisting of white, black and grey boxes to demonstrate the tight coupling of phenomena and paradigms in the field and discusses deconstructing OB’s white box to encourage data-driven phenomena to coexist in the spatial framework. Design/methodology/approach A scoping literature review was conducted to offer a preliminary assessment of technology-oriented research currently occurring in OB. Findings The literature search revealed two findings. First, the number of published papers on emerging technologies in top management journals has been increasing at a steady pace. Second, various theoretical perspectives at the micro- and macro- organizational level have been used so far for conducting technology-oriented research. Originality/value By conducting a scoping review of emerging technologies research in OB literature, this paper reveals a conceptual black box relating to technology-oriented research. The essay advocates for loosening OB’s tightly coupled white box to incorporate emerging technologies both as a phenomenon and as data analytical techniques.


2021 ◽  
Author(s):  
Kelly H. Oh ◽  
Mia Krout ◽  
Janet E. Richmond ◽  
Hongkyun Kim

AbstractPresynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners.Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.


2016 ◽  
Vol 213 (6) ◽  
pp. 613-615 ◽  
Author(s):  
Camilla Raiborg ◽  
Harald Stenmark

Lysosomes fuse with the plasma membrane to help repair membrane lesions, but how they are positioned close to these lesions is not fully understood. Now, Encarnação et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201511093) demonstrate that the lysosomal GTPase Rab3a and its effectors orchestrate lysosome positioning and plasma membrane repair.


1997 ◽  
Vol 110 (1) ◽  
pp. 11-21 ◽  
Author(s):  
M. Glogauer ◽  
P. Arora ◽  
G. Yao ◽  
I. Sokholov ◽  
J. Ferrier ◽  
...  

The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.


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